The present invention relates to novel calixcrown derivatives, a process for the preparation thereof, a self-assembled monolayer prepared by using the same and a process for immobilizing a protein monolayer by using the self-assembled monolayer of the novel calixcrown derivatives. In particular, the present invention relates to novel calixcrown derivatives having thiol functions indispensable for the preparation of a self-assembled monolayer of said calixcrown derivatives, which can be applied for immobilization of proteins by multiple ionic recognition. The present invention further relates to a process for the preparation of said novel calixcrown derivatives, a self-assembled monolayer prepared by applying said calixcrown derivatives to a gold substrate or related metal substrate and a process for immobilizing a protein monolayer by using the self-assembled monolayer.
The immobilization of enzymes, antigens, antibodies and the like on solid carriers has become one of the most basic techniques in biotechnology or in protein research, such as immunochemistry and enzyme chemistry. For example, the enzyme linked immunosorbentassay (ELISA) is a technique that has been widely used in biotechnology for the assay of a particular protein or specific proteins causing a certain disease in experimental or clinical laboratories. Assay kits of such ELISA are commercially available in the market. More recently, development of protein chips, which require improved methods of protein immobilization on a solid matrix, is of a great concern in the field of biotechnology for the further advancement of proteomics research in the post-genomic era.
Previously, the immobilization of proteins such as antigens, antibodies or enzymes has been commonly practiced by physical adsorption of said proteins on a high molecular weight biopolymers such as various derivatives of collagen, dextran or cellulose. Covalent bonding between proteins and carrier surface by chemical reaction has been also widely used as a method for protein immobilization. The protein immobilization method by a xe2x80x9cSandwichxe2x80x9d technique (triple-molecular layer) has been disclosed in literature [Science, 1993, Vol. 262, ppl706-1708], which describes a chemical bonding method by the biotin-avidin (or streptoavidin) interaction between proteins and carrier surface. That is, biotin is attached to the carrier surface and subsequently avidin or steptoavidin is linked thereto. Finally, proteins linked with biotin can be immobilized on said chemically modified carrier surface.
However, numerous problems are present in the various methods of protein immobilization described above. The physical adsorption method as well as the methods of covalent bonding and the biotin-avidine binding, which have recently been used, do bear problems as follows.
1. Density
The most critical problem of the protein immobilization method used in the past has been noted as that the amount of protein immobilized on the surface of a substrate is extremely small. When the density of a protein to be immobilized on a carrier surface is low, other proteins may form non-specific binding. It is thus necessary to carry out chemical treatment for the carrier surface so as to eliminate the undesired proteins bound to the carrier surface. However, such a chemical treatment may cause inactivation or denaturation of the immobilized protein molecule. In addition, even if a specific target protein is immobilized successfully onto the surface of a carrier, only an extremely small amount of the protein can be captured and consequently, it is often required that the assay result be further confirmed by various assay methods. It is also noted that the more the amount of proteins is immobilized on an unit area on the surface of a carrier, the easier the assay process is. In this regard, many studies have been carried for the development of methods for a single molecular layer of proteins with the maximum amount immobilized on a carrier surface. A satisfactory result, however, is yet to be achieved.
2. Activity
In prior methods for protein immobilization by either chemical bonding or physical adsorption on surface of a carrier, the activity of an immobilized protein could be decreased in comparison with the free protein in a solution. It has been known as the reason that an immobilized protein on a solid carrier could lose its activity due to conformational changes or denaturation of the protein especially around its active site as it binds tightly to the carrier surface via physisorption or chemical binding.
3. Orientation
In prior methods for protein immobilization on surface of a carrier, an active site of the protein may become essentially oriented toward the carrier surface in such a way that the active site is masked and thus the activity of the protein becomes lost. Such orientation of the protein is also a serious problem in procedures of protein immobilization. It is known that such phenomena is occurred in almost half of the immobilized proteins.
An object of the present invention is to provide calixcrown derivatives having two recognition sites essential as a molecular linker applicable for protein immobilization onto the surface of a carrier, one of which can recognize the ammonium residue of a protein while the other can bind tightly to the surface of a carrier; said calixcrown derivatives are active for the formation of a self-assembled monolayer, which is useful for a- process of protein immobilization solving the problems of prior processes. Another object of the present invention is to provide a process for the preparation of said novel calixcrown derivatives.
A further object of the present invention is to provide a self-assembled monolayer of calixcrown derivatives by binding said calixcrown derivatives on a gold substrate or related metal substrate; said monolayer of calixcrown derivatives can be used for high-density protein immobilization on a monolayer, the surface of which can allow to interact with all kinds of other proteins in a test solution thereof without any additional complicated procedures.
A further object of the present invention is to provide a immobilization process for protein molecules such as antigens, antibodies or enzymes of which the molecular weight are respectively not less than 20,000 D (20KD) on said self-assembled monolayer.
A further object of the present invention is to provide an immobilization process for said protein molecules by applying the compounds of formulae 1 to 3 of the present invention onto inorganic or organic solid substrate such as gold, silver, glass, silicon, polystyrene, polycarbonate, etc. for the preparation of protein chip, diagnostic kit, protein separation pack, etc.